An ADAMTS3 missense variant is associated with Norwich Terrier upper airway syndrome.

In flat-faced dog breeds, air resistance caused by skull conformation is believed to be a major determinant of Brachycephalic Obstructive Airway Syndrome (BOAS). The clinical presentation of BOAS is heterogeneous, suggesting determinants independent of skull conformation contribute to airway disease. Norwich Terriers, a mesocephalic breed, are predisposed to Upper Airway Syndrome (UAS), a disease whose pathological features overlap with BOAS. Our health screening clinic examined and scored the airways of 401 Norwich terriers by laryngoscopy. Genome-wide association analyses of UAS-related pathologies revealed a genetic association on canine chromosome 13 (rs9043975, p = 7.79x10-16). Whole genome resequencing was used to identify causal variant(s) within a 414 kb critical interval. This approach highlighted an error in the CanFam3.1 dog assembly, which when resolved, led to the discovery of a c.2786G>A missense variant in exon 20 of the positional candidate gene, ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). In addition to segregating with UAS amongst Norwich Terriers, the ADAMTS3 c.2786G>A risk allele frequency was enriched among the BOAS-susceptible French and (English) Bulldogs. Previous studies indicate that ADAMTS3 loss of function results in lymphoedema. Our results suggest a new paradigm in the understanding of canine upper airway disease aetiology: airway oedema caused by disruption of ADAMTS3 predisposes dogs to respiratory obstruction. These findings will enhance breeding practices and could refine the prognostics of surgical interventions that are often used to treat airway obstruction.

A de novo variant in the ASPRV1 gene in a dog with ichthyosis.

Ichthyoses are a heterogeneous group of inherited cornification disorders characterized by generalized dry skin, scaling and/or hyperkeratosis. Ichthyosis vulgaris is the most common form of ichthyosis in humans and caused by genetic variants in the FLG gene encoding filaggrin. Filaggrin is a key player in the formation of the stratum corneum, the uppermost layer of the epidermis and therefore crucial for barrier function. During terminal differentiation of keratinocytes, the precursor profilaggrin is cleaved by several proteases into filaggrin monomers and eventually processed into free amino acids contributing to the hydration of the cornified layer. We studied a German Shepherd dog with a novel form of ichthyosis. Comparing the genome sequence of the affected dog with 288 genomes from genetically diverse non-affected dogs we identified a private heterozygous variant in the ASPRV1 gene encoding "aspartic peptidase, retroviral-like 1", which is also known as skin aspartic protease (SASPase). The variant was absent in both parents and therefore due to a de novo mutation event. It was a missense variant, c.1052T>C, affecting a conserved residue close to an autoprocessing cleavage site, p.(Leu351Pro). ASPRV1 encodes a retroviral-like protease involved in profilaggrin-to-filaggrin processing. By immunofluorescence staining we showed that the filaggrin expression pattern was altered in the affected dog. Thus, our findings provide strong evidence that the identified de novo variant is causative for the ichthyosis in the affected dog and that ASPRV1 plays an essential role in skin barrier formation. ASPRV1 is thus a novel candidate gene for unexplained human forms of ichthyoses.

Generalized myoclonic epilepsy with photosensitivity in juvenile dogs caused by a defective DIRAS family GTPase 1.

The clinical and electroencephalographic features of a canine generalized myoclonic epilepsy with photosensitivity and onset in young Rhodesian Ridgeback dogs (6 wk to 18 mo) are described. A fully penetrant recessive 4-bp deletion was identified in the DIRAS family GTPase 1 (DIRAS1) gene with an altered expression pattern of DIRAS1 protein in the affected brain. This neuronal DIRAS1 gene with a proposed role in cholinergic transmission provides not only a candidate for human myoclonic epilepsy but also insights into the disease etiology, while establishing a spontaneous model for future intervention studies and functional characterization.

Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

LGI2 truncation causes a remitting focal epilepsy in dogs.

One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2-10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission.

YARS2 Missense Variant in Belgian Shepherd Dogs with Cardiomyopathy and Juvenile Mortality.

Dog puppy loss by the age of six to eight weeks after normal development is relatively uncommon. Necropsy findings in two spontaneously deceased Belgian Shepherd puppies indicated an abnormal accumulation of material in several organs. A third deceased puppy exhibited mild signs of an inflammation in the central nervous system and an enteritis. The puppies were closely related, raising the suspicion of a genetic cause. Pedigree analysis suggested a monogenic autosomal recessive inheritance. Combined linkage and homozygosity mapping assigned the most likely position of a potential genetic defect to 13 genome segments totaling 82 Mb. The genome of an affected puppy was sequenced and compared to 645 control genomes. Three private protein changing variants were found in the linked and homozygous regions. Targeted genotyping in 96 Belgian Shepherd dogs excluded two of these variants. The remaining variant, YARS2:1054G>A or p.Glu352Lys, was perfectly associated with the phenotype in a cohort of 474 Belgian Shepherd dogs.YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase 2 and the predicted amino acid change replaces a negatively charged and evolutionary conserved glutamate at the surface of the tRNA binding domain of YARS2 with a positively charged lysine. Human patients with loss-of-function variants in YARS2 suffer from myopathy, lactic acidosis, and sideroblastic anemia 2, a disease with clinical similarities to the phenotype of the studied dogs. The carrier frequency was 27.2% in the tested Belgian Shepherd dogs. Our data suggest YARS2:1054G>A as the candidate causative variant for the observed juvenile mortality.

A Missense Variant in SCN8A in Alpine Dachsbracke Dogs Affected by Spinocerebellar Ataxia.

Spinocerebellar ataxias is an umbrella term for clinically- and neuropathologically-heterogeneous early-onset hereditary neurodegenerative diseases affecting several dog breeds. The purpose of this study is to identify the causative genetic variant associated with ataxia, tremor, and loss of balance in Alpine Dachsbracke dogs. We investigated two related litters in which four cases were reported. Neuropathology of two dogs revealed spongy degeneration associated with axonal degeneration. Combined genetic linkage and autozygosity analyses in four cases and eight related controls showed one critical disease-associated interval on chromosomes 27. Private whole-genome sequence variants of one ataxia case against 600 unrelated controls revealed one protein-changing variant within the critical interval in the SCN8A gene (c.4898G>T; p.Gly1633Val). Perfect segregation with the phenotype was confirmed by genotyping >200 Alpine Dachsbracke dogs. SCN8A encodes a voltage-gated sodium channel and the missense variant was predicted deleterious by three different in silico prediction tools. Pathogenic variants in SCN8A were previously reported in humans with ataxia, pancerebellar atrophy, and cognitive disability. Furthermore, cerebellar ataxia syndrome in the 'jolting' mutant mice is caused by a missense variant in Scn8a. Therefore, we considered the SCN8A:c.4898G>T variant to be the most likely cause for recessively inherited spinocerebellar ataxia in Alpine Dachsbracke dogs.

A SINE Insertion in ATP1B2 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA2).

Spongy degeneration with cerebellar ataxia (SDCA) is a genetically heterogeneous neurodegenerative disorder with autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. Using a combined linkage and homozygosity mapping approach we identified an ∼10.6 Mb critical interval on chromosome 5 in a Malinois family with four puppies affected by cerebellar dysfunction. Visual inspection of the 10.6 Mb interval in whole-genome sequencing data from one affected puppy revealed a 227 bp SINE insertion into the ATP1B2 gene encoding the ß2 subunit of the Na+/K+-ATPase holoenzyme (ATP1B2:c.130_131insLT796559.1:g.50_276). The SINE insertion caused aberrant RNA splicing. Immunohistochemistry suggested a reduction of ATP1B2 protein expression in the central nervous system of affected puppies. Atp1b2 knockout mice had previously been reported to show clinical and neurohistopathological findings similar to the affected Malinois puppies. Therefore, we consider ATP1B2:c.130_131ins227 the most likely candidate causative variant for a second subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia subtype 2 (SDCA2). Our study further elucidates the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population in Malinois and the other varieties of the Belgian Shepherd breed. ATP1B2 thus represents another candidate gene for human inherited cerebellar ataxias, and SDCA2-affected Malinois puppies may serve as a naturally occurring animal model for this disorder.

A Missense Variant in KCNJ10 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA1).

Spongy degeneration with cerebellar ataxia (SDCA) is a severe neurodegenerative disease with monogenic autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. We performed a genetic investigation in six families and seven isolated cases of Malinois dogs with signs of cerebellar dysfunction. Linkage analysis revealed an unexpected genetic heterogeneity within the studied cases. The affected dogs from four families and one isolated case shared a ∼1.4 Mb common homozygous haplotype segment on chromosome 38. Whole genome sequence analysis of three affected and 140 control dogs revealed a missense variant in the KCNJ10 gene encoding a potassium channel (c.986T>C; p.Leu329Pro). Pathogenic variants in KCNJ10 were reported previously in humans, mice, and dogs with neurological phenotypes. Therefore, we consider KCNJ10:c.986T>C the most likely candidate causative variant for one subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia 1 (SDCA1). However, our study also comprised samples from 12 Malinois dogs with cerebellar dysfunction which were not homozygous for this variant, suggesting a different genetic basis in these dogs. A retrospective detailed clinical and histopathological analysis revealed subtle neuropathological differences with respect to SDCA1-affected dogs. Thus, our study highlights the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population. These dogs represent an animal model for the human EAST syndrome.

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses.

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.

A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12.

Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds--the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10(-11)). Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018-5,081,823 bp), tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8) and does not extend to the dog leukocyte antigen (DLA) class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.

A COL11A2 mutation in Labrador retrievers with mild disproportionate dwarfism.

We describe a mild form of disproportionate dwarfism in Labrador Retrievers, which is not associated with any obvious health problems such as secondary arthrosis. We designate this phenotype as skeletal dysplasia 2 (SD2). It is inherited as a monogenic autosomal recessive trait with incomplete penetrance primarily in working lines of the Labrador Retriever breed. Using 23 cases and 37 controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 4.44 Mb interval on chromosome 12. We re-sequenced the genome of one affected dog at 30x coverage and detected 92 non-synonymous variants in the critical interval. Only two of these variants, located in the lymphotoxin A (LTA) and collagen alpha-2(XI) chain gene (COL11A2), respectively, were perfectly associated with the trait. Previously described COL11A2 variants in humans or mice lead to skeletal dysplasias and/or deafness. The dog variant associated with disproportionate dwarfism, COL11A2:c.143G>C or p.R48P, probably has only a minor effect on collagen XI function, which might explain the comparatively mild phenotype seen in our study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of COL11A2 mutations. We speculate that non-pathogenic COL11A2 variants might even contribute to the heritable variation in height.